Current Research Journal of Biological Sciences 1(3): 139-143, 2009
ISSN: 2041-0778
© M axwell Scientific Organization, 2009
Submitted Date: July 24, 2009
Accepted Date: August 19, 2009
Published Date: October 20, 2009
Bioactive Studies of Mangifera indica aganist Bacteria Isolated from Urine Samples
1
1
S. Sowmiy a, 1 P. Soundarapandian and 2 S. Rajan
Centre of Advanced Stud y in Marine Biology , Annam alai University Parangip ettai
608 502, Tamil Nadu, India.
2
Srimad Andavan Arts and Science College, Tiruchirapalli-620 005
Abstract: Urinary tract infections are the second most common type of infection in the w orld. It is usu ally
caused by bacteria such as Escherichia co li, Pseudomonas aeruginosa, Staphylococcus aureus, Kleb isella
pneumoniae, Streptococcus pyogens etc. Bacteriuria, Pyrex ia, Uretheritis, Pyuria, Haematuria are the urinary
tract infection, comm only caused by b acteria. Seed ke rnel of Mangifera indica which has antiba cterial,
antidiarrhoeal, antioxidant and antiviral activity against the uropathogens isolated from clinical samples. Urine
samples were collected from government hospital at Srirangam. The samples w ere subjected to macroscopic,
microscopic, and culturing by selective and differential media and biochemical test to identify the pathogens.
Seed kernels of M. indica were extracted by using water and ethanol. Antibacterial activity of M. indica
aqueous and ethanol was studied by disc diffusion method, which shows best activity against the clinical
isolates at 800 :g conc entration (19mm ). St. aureus is strongly inhibited by the extract. MIC w as performed
by using agar dilution m ethod . It was found to be between 3 00m g/ml to 750mg/m l. Phytochemical studies
revealed that the presence of Tannin and Steroid. If we develop a good drug from this plant it may be better for
human health.
Key w ords: Mangifera indica, seed kernels, Staphylococcus aureus and urinary tract infections
INTRODUCTION
Urinary tract infections are serious health problem
affecting millions of people each year. Urinary tract
infections account for about 8.3 million doctor visit each
year. One woman in five develops a UTI during her
lifetime. Escherichia coli, Klbesiella, Streptococcus
pyogens, St. faeca lis, Pseu dom onas vulgaris, P. faecalis
are the bacteria responsible for urinary tract infection.
Comm only used antibiotics for the treatmen t of urinary
tract infection are Clindamycin, Vancomycin, Bacitracin,
Ampicillin, Chloramphenicol, and Erythromycin. These
antibiotics may reduce the burden but it has its own side
effects. To overco me the prob lems assoc iated w ith
antibiotic treatment, people turned traditional medicine
like Ayurveda, Siddha, unani, Homeopathy and herbal
medicines. About 80% of world populations rely on
herbal medicine for primary health care (WHO /WPRO,
1998). Thousands of medicinal plants ava ilable locally are
used to treat urinary tract infection. Som e of the plants
leaves of Punica grantaum, seed kerne l Mangifera indica,
whole plant of Tridax procumbans, whole plant of Aerva
lanna ta were already used to control UTI. Based on
variou s usag es in the present study we have chosen seed
kernel of M. indica to scree n antibacterial activity,
antidiarrhoeal activity and its phytochemistry.
MATERIALS AND METHODS
Urine samples were collected from clinically
diagnosed urinary infectious patients admitted in
Government hospital, at Srirangam, Tamil Nadu, India.
Samples were collected for three months from
(January-M arch, 2007 ).
Urine samples were collected from clinically
diagnosed cases of Urinary tract infection. Seed kernel of
M. indica was the plant materials chosen for anti-bacterial
activity studies. E. coli, S. aureus and St. pyogens were
isolated from the urinary tract infectious patients we re
used as test org anism .
Both broad spectrum and narrow spectrum antibiotics
were used to assess sensitivity pattern of the clinical
isolates i.e., E. coli, S. aureus and St. pyogens. Antibiotics
like Azithromycin (15:g), Carbenicillin (100:g),
Ce furox im e ( 1 0: g ) , C h l o ra m p h e nic ol (30: g),
Doxycycline hydrochloride (30:g), Minocycline (30:g),
Nalidixicac id (30:g), Rifamy cin (5:g), Vanco myc in
(30:g), Clarithromycin (15:g), Trimethop rim (5:g),
Spectinom ycin (100:g), Amoxyclave (30:g), and
tetracycline (30:g), Co-trimoxazole, Clinadamycin,
Erythromycin and Bacitracin, were used to see the
sensitivity pattern of enteric pathogens.
Source of Plant M ateria l: Good quality Seed kernel of
M .indica was collected locally and identified.
Urine collection: Urine samples were collected in a wide
mouthed container (H i-med ia) from clinically diagnosed
patients. After collection of samples, the containers w ere
closed tightly to avoid any leakage during transportation,
(Koneman et al., 1994). V arious types of processing w ere
done to find out the correct etiological agent of the
disease. Processing varies in accordance with the aim of
the study an d the pathogens look ed for.
Corresponding Author: S. Rajan, Srimad Andavan Arts and Science College, Tiruchirapalli-620 005, India
139
Curr. Res. J. Biol. Sci., 1(3): 139-143, 2009
E. coli is one of the gram nega tive bacteria. It belongs to
the family Enterobacteriaceae. Enrichment, differential
and selective media were used to isolate E. coli.
Staphylococcus and Streptococcus is the gram-positive
organism. Differential and selective media w ere used to
isolate these organisms.
A loop fu ll of urine was taken and inoculated on
Macconkey agar and Blood agar with antibiotics disc for
differentiation of gram positive organism and was
incubated at 37 ºC for 24 h under aerobic condition. Pink
color colony and hemolytic colonies was selected from
Macconkey agar and B lood a gar an d it was subjected to
sub culturing for further screening p roced ure. A ll of them
were incubated aerobically at 37 ºC for 24 h and were
looked for specific colony m orphology , which w ould
confirm the isolation of E. coli, S. aureus and St. pyogens.
The growth was observed in selective and differential
medium and the results were tabulated.
E. coli, S. aureus and St. pyogens was identified by
making use of bioch emical tests in addition to its gro wth
characters on nutrient agar and microscopic analysis.
Selected colonies from selective and differential
med ia were subjected to microscopy, microscopy and
biochemical tests for identification.
Microscopic observations like size, shape and
motility reveal the availability of different morphological
characters amo ng m icroorg anism s. Simple staining, gram
staining and hanging drop methods were done to look for
their shape, gram s nature and motility of the isolate
respectively (H enry, 1994).
Gram staining was performed to look for the grams
nature of the isolate. A purple coloured cell retains grams
crystal violet and was called gram positive bacterium.
Pink coloured cells lost primary stain and picked up
safranin color and were called as gram negative
bacterium.
Bacteria were motile by their flagella. The number
and location of which vary among different species.
M otility can be observed directly by hanging drop
technique that is by placing a drop of culture on a
micro scop ic slide and looked under microscope by
keeping them inverted.
Physiological and metabolic characteristics of the
microorganisms were assessed through biochemical tests.
These characteristics are very useful because they are
directly related to the nature and activity of microbial
enzymes and transpo rt proteins. Analysis of these
characteristics provides an indirect comparison of
microbial geno mes. The follow ing tests were used to
characterize microbial enzymes and proteins.
Indole test, Methyl red test (MR) Vo ges Proskauer
test (VP), Citrate utilization test (C), Urea se production
test (U), Nitrate reduction test (N), Cytochrome ox idase
activity , Catalase test were tested by adopting the method
of Koneman et al. (1998)
suspension and spread onto one half of a blood agar plate.
A 100 mg Furazolidone disc is placed aseptically in the
center of the inoculated area. The plate is incubated at
35ºC for 18-24 h and observed for zone formation.
Staphylococci are inhibited by Furazolidone and shows
zone of 15 mm. The coagulase negative Staph yloco cci are
resistant to Furazolidone.
Resistant to Bacitracin: The Bacitracin disk was used
for the presumptive identification of group A, Betahem olytic streptococci on Muller-Hinton Agar or Blood
agar plate. After incub ation, Staphylococcus was resistant
to Bacitracin.
Slide coagu lase test: The tests were performed by placing
two drops of sterile water or saline on a slide. The well
isolated colon ies of organism are emu lsified in the liquid
within each of the circle. A drop of coag ulase plasm a is
added to one of the suspension and mixed with a wooden
applicator stick; similarly, a drop of w ater or sa line is
added and mixed in the other suspension as control. The
suspension is then observed for agglutination.
DN ase test: St.aureus strain procedure weak or equivocal
tube coagulase reaction, which reaction may be helpful to
perform other test. S. aureus produce DNase and
therm ostable endonuclease. These hydrolyze nucleic acid
change the colour formation of metachromatic dye
toluidine blue O into pink. After incubation period for 24
h at 37º C it indicates the hydrolysis of the DNA.
Hydrolysis of L-pyrrolindonyl B-naphthylamide
(PYR ): PYR hydrolysis is a presumptive test for both
groups A and D Enteroco ccal and Streptococci. This test
is highly sensitive. It replaces the Bacitracin test and the
salt tolerance test for group A , Streptococ ci.
Bile-E sculin test: To hydrolyze esculin in the presence of
40% bile is used for the presumptive identification of
group D Streptococci and Enterococcus sp. Th is test is
generally perform ed on an agar slant or in a plate that
contain the bile-esculin medium. Any blackening of the
agar in the plate indica tes a po sitive result.
Salt Tolera nce T est (6.5% Nac l broth): The salt
tolerance test was based on the ability of a n organism to
grow in 6.5% NaCl, separates the Streptococ cal sp. The
organism to be identified is inoculated into an infusion
based agar or broth containing 6.5% NaCl. After
incubation, the medium is observed for the growth.
Susceptibility to Ethyl hydrocupreine hydrochloride
(optochin): To perform the test, a few colonies are sub
cultured to a Blood agar plate and are streaked as lawn.
An optochin disc is placed on the inoculum and the plate
is incubated a t 35ºC in 5% CO 2 . A zone of 14 mm or
greater around the 6mm indicates the susceptibility to
optochin.
Inhibition by Furazolidone: It was performed by
preparing a suspension of the test organism in distilled
water or broth equiva lent to the test orga nism in
140
Curr. Res. J. Biol. Sci., 1(3): 139-143, 2009
Table 1: Samples collection details and categories based on sex
Total No. of samples
M ale
Fem ale
25
8
17
Bile Solubility test: The test can be perform ed on a broth
or saline suspe nsion of the organism or directly on a plate.
In the tube test, clea ring of the 10% deox ycholate
suspension after the inoculation of unknown organism and
incubation for 3 h indicates lysis of the bacterial cells. For
the plate test, a drop of 2% sodium deoxycholate is placed
directly on a few colonies of the organism and incubated
at 35ºC without inverting for 30 min. The colonies will
lyses and disappears leaving only the area of haemolysis.
Tab le 3:
S No
1
2
3
Urinary Tract and produced severe Urinary Tract
Infection. Uropathogens are responsible for majority of
human infection. Bacteria are the major flora that causes
severe Urinary Tract infection then other microbial
groups. Culture media like Blood Agar, Macconkey Agar
are primarily used for the recovery of uropathogens.
Totally 10 E. coli, uropathogens are isolated from the
clinical samples (55%) followed by S. aureus (5 numbers
and 28% ) St. pyogens (3 numbers and 17%).Various
biochemical test were used for the diagnosis of
uropathogens (Ta ble 3).
Antibiotics are medicines that fight against infections
caused by bacteria. Because antibiotics are used a lot
something used in appropriately, antibiotic resistant is
becoming a common problem in many parts of the world.
RESULTS
W e have collected 25 samples from government
hospital at Srirangam. All these samples were collected
from clinically diagnostic cases. Samples were categories
based on sex. A bout 68% of our samples were from
female and it is double times higher than males (32%)
(Table 1).
All the clinical samples were subjected to
microbiological examination. In the present study
bacterial etiology was noted 18 samples out of 25% (72%)
(Table 2).
Bacteria are the group of microorganism that belongs
to the group prokaryotes that multiplied with in the
Table 2: Identification of Bacteria from U rinary tract isolates
M edia
E. C oli
EMB
Metallic Sheen Colour
XLD
Yellow colour
Macconk ey
LF
Blood Ag ar
Baired Parker Agar
NM
M
NP
+
Van - R
Bac – Sen
Streptococcus P yogens
Staphylococcus aureus
E. C oli
A /A
Table
S .N o
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
2: (Continue)
Test
US1
Gram staining +
Shape
Cocci
M otility
N/M
Indole test
Methyl red
test
+
Vogespros
kaur test
Citrate
utilization test N P
Urease test
NP
TSI test
NP
H2S
NP
Gas
NP
Nitrate
reduction test N P
Catala se tests Oxidase test +
Carbohydrate test
Glucose
NP
Maltose
NP
Sucrose
NP
Incidence of bacterial etiology selected for antib acteria l
screening
Isolated organisms
Numb er of isolates
E.co li
10
Staphylococcus aureus
5
Streptococcus pyogens
3
Streptococcus Pyogens
Staphylococcus aureus
Non - Metallic Sheen Colour
Non - Metallic Sheen Colour
colourless
colourless
NLF
NLF
$- Hemolysis Van – R
$- Hemolysis Bac – Sen
Black Colour Vanc – R
-No n M otile
-Motile
-Not Performed
-Positive
-Negative
-Vancomycin Resistant
-Bacitracin Sensitive
-U S1 , US 14 , US 15 ,
- US3, US7, US8, US9, US16
- US2, US4, US5, US6, US10, US11,US12, US13, US17, US18
-Ac id / A cid
US2
Rod
M
+
US3
+
Cocci
N/M
-
US4
Rod
M
+
US5
Rod
M
+
US6
Rod
M
+
U S7
+
Cocci
N/M
-
U S8
+
Cocci
N/M
-
U S9
+
Cocci
N/M
-
US10
Rod
M
+
US11
Rod
M
+
US12
Rod
M
+
US13
Rod
M
+
US14
+
Cocci
N/M
-
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
-
-
-
-
-
-
-
-
-
A/A
+
NP
NP
NP
NP
NP
A/A
+
A/A
+
A/A
+
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
A/A
+
A/A
+
A/A
+
+
+
-
NP
+
-
+
+
-
+
+
-
+
+
-
NP
+
-
NP
+
-
NP
+
-
+
+
-
+
+
-
+
+
+
NP
NP
NP
+
+
+
+
+
+
+
+
+
NP
NP
NP
NP
NP
NP
NP
NP
NP
+
+
+
+
+
+
141
U S15
+
Cocci
N/M
-
US16
+
cocci
N/M
-
US17
Rod
M
+
US18
Rod
M
+
+
+
+
+
-
-
-
-
-
A/A
+
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
A/A
+
A/A
+
+
+
-
+
+
-
NP
+
NP
+
NP
+
-
+
+
-
+
+
-
+
+
+
+
+
+
NP
NP
NP
NP
NP
NP
NP
NP
NP
+
+
+
+
+
+
Curr. Res. J. Biol. Sci., 1(3): 139-143, 2009
Table 4: A ntibiotic Sensitivity Assay P atterns of Clinical Isolates
S .N o
Antibiotics
Eco li
-----------------------------S
R
1.
Deoxycline hydrochloride
50%
50%
2.
Rifa mp icin
40%
60%
3.
Az ithrom ycin
30%
70%
4.
Sp ectin om ycin
20%
80%
5.
Trimethoprin
70%
30%
6.
Cefaroxine
60%
40%
7.
M inoc yclin
70%
30%
8.
Cla rithrom icin
20%
80%
9.
Va nco my cin
50%
50%
10.
Ca rben icillin
30%
70%
11.
Ery throm ycin
40%
60%
12.
Chloromphenicol
70%
30%
13.
Trim oxa zole
50%
50%
14.
Clin dam ycin
20%
80%
15.
Amoxyclave
60%
40%
16.
Tertracuicline
70%
30%
17.
Nalidicin
60%
40%
18
Ba citracin
60%
40%
Table 5: A ntibacterial activity of M. indica against enteric pathogens
S .N o
Ex tracts
Zone of inhibition
E. co li.(800:g/disc)(mm)
1
Aqueous extract
13
2
Ethanol extract
8
3
Positive control
14
4
Negative control
Nil
Tab le 6:
Phytochem istry of M. indica extra cts
S No
Tes ts
Aqueous extract
1
Ca rbo hyd rate
+
2
Pro tein
3
Alkaloids
+
4
Glycosides
5
Terpenoids
6
Flavanoids
7
Tanins
+
8
Saponins
+
9
Steroids
10
Starch
+
+ - Positive, - - Negative
Streptococcus pyogens
------------------------------S
R
80%
20%
60%
40%
40%
60%
20%
80%
40%
60%
60%
40%
80%
20%
60%
40%
40%
60%
20%
80%
80%
20%
60%
40%
40%
60%
80%
20%
60%
40%
40%
60%
20%
80%
80%
20%
Zone of inhibition Staphylococcus
aureus(800:g/disc)(mm)
19
19
22
Nil
Staphylococcus aureus
-------------------------------S
R
33%
67%
67%
33%
33%
67%
67%
33%
67%
33%
33%
67%
67%
33%
33%
67%
33%
67%
67%
33%
33%
67%
67%
33%
33%
67%
67%
33%
33%
67%
33%
67%
67%
33%
33%
67%
Zone of inhibition Streptococcus
pyogen s (800:g/disc)(mm)
11
12
27
Nil
MIC of M.indica extract was studie d by using Agar
Dilution Method , W hich also sho ws inhibitory activity
against all the pathoge ns. A phytoche mical constituent are
the principle source of the medicinal plant and is
respo nsible for various biological activities. Tannis are
the major component found in b oth the extract of M.
indica (Table 6).
Ethanol extract
+
+
+
+
+
DISCUSSION
Urinary Tract Infection are not considered as
outbreak diseases bu t it security is high in human . In
general com mun ity acquired Urinary Tract Infection
occurs mostly in women. Bladder infections are 14 times
more common in females than ma les. Uropathoge ns is
easily entered into fem ale Urinary T ract and cau se severe
life threading info (An nonym ous, 2001).
Our results also revealed the same about 68% of
samples were collected from female cases which is two
times higher than male. This may be due to shorter
urethra. Hormones and ch emical barriers prese nt in
females urethra.
Randrianirina (2007) also reported that higher
incidence of female Urinary Tract Infection (75% ) than in
male (25%).Multiple factors have probably lead to the
emergence and spread of Urinary Tract Infection.
M ajority of Urinary Tract Infection are caused by
uropathog enic bacteria about 72% of Urinary Tract
Infection are due to bacteria is noted in the present study.
This was also supported by David et al. (2005). He
reported that comm on U rinary Tract Infection is caused
by bacteria. It means presence of bacteria in urine. Among
fact E. coli shows higher incidence (Elmanana et al.,
To know the antibiotic resistant pattern of our isolates we
conducted antibiotics assay by making the use of Disk
Diffusion m ethod .
About 18 commonly used antibiotics are subjected
for the study. Out current report showed that maximum
number of isolates were sensitive to new generation
a n t i b io t i c s lik e c a r b e n z il i n , C h lo ram phe nic ol,
Tetracycline etc. Abou t 75% of E.coli, were resistant to
amzoxyclave and 80% of S. aureus to carbenzilin and
more than 60% of St. pyogens to Deoxycycline,
Rifamp icin, carbenzilin, Erythrom ycin, etc. (Table 4).
M. indica is a medicinal tree com mon ly called as ma
in Tam il and m ango in English. All parts of this tree are
com mon ly used in traditional system of medicine. Seed
kernel of M. indica is selected for the study due to its
common med icinal usage. The seed kernel is collected
from local market of Srirangam and processed to extract
its content by making use of water and ethanol. Both of
these extract show ed inh ibitory activity against all the
pathogens tested. E thano l extract show ed be tter activity
then water extract (Table 5).
142
Curr. Res. J. Biol. Sci., 1(3): 139-143, 2009
2006) isolated 42% of E. coli followed by minimum
number of other pathogens.
Ronald (2003) repo rted that Urinary Tract Infection
associated with microbial etiology is reasona bly
consistence. E. coli, uropathogens 80% followed by
Klebsiella, Enterobacter, Staphylococcus, and Proteus
etc. is isolated from the urinary tract infection.
Our present study also revealed the presence of 55%
of E. coli followed by 28% S. aureus and 17% of St.
pyogens. Ronald (2003) indicated that about 10 – 15% of
Urinary Tract Infection is due to staphylococcus.
Kiffer et al., (2007) isolated 13% of gram positive
cocci from the Urinary Tract infected individual from
urban areas. We also conducted the study from urban
based com mun ity environm ent.
Antibiotic resistance is a major problem of clinical
peoples. It is an evaluation process of microorganisms.
Majority of Uropathogens developed resistant against
second and third generation cephaloporins and other
com mon ly used drugs. W e hav e used 18 antibiotics in the
present study. Out of large number of isolates are resistant
to multiple drugs and these microo rganisms are
considered as multi drug resistant patho gens. Resistant to
antimicrobial was extremely allomining. Lee et al. (2007)
showed that E. coli resistant to amo xicillin was reached
97.9% to piperacillin 78.3% to Doxycycline, 90% to
sulfa-methoxazole 63.9% and to cefaclor 42%.
Over 20,000 p ractioners of Indian system of medicine
in the oral and co dified stream uses m edicinal plants in
preventive, prom otive and cu rative application in T amil
Nadu. M. indica and its parts used to cure various
purposes. The seed of M. indica is reported in traditional
medicine as a cure for vomiting, dysentery and burning.
Paste is made from mango seed (kernel), honey and
camphor and applied over vagina in order to make the
vagina co ntracted and firm (Sharma , 1996).
Seed kernel of aqueous and ethanolic extract
inhibited the grow th of S .aureus, P .vulga nis (Sairam
et al., 2003). Our aqueous and ethanol extract showed
good antibacterial activity, against E. coli, S. aureus and
St. pyogens. Both these e xtract trac t show ed be st activity
against S. aureus at 800 :g concentration (19 mm ). This
antibacterial activity of M. indica may be due to specific
phytochemical compon ents.
Phytochemical compounds are the key factor to
perform biolog ical activities like anti bacterial activity,
antifun gal, antiprotozoan, antioxidant etc. Tannins are
respo nsible for phytochemical activity. Dried mango seed
contain 15% tannin served as astringent in cases of
diarrhea, dysentery, uretheritis etc. Toxic com ponents are
not de tected in seed kerne l.
They are the safe source of antioxidant. We also
estimated tannins from aqueous ethanolic extract of seed
kernel of M. indica. Saponins, alka loids starch are also
present in these extract.
Medicinal plants are the chief source of medicine and
are used for the trea tmen t of varies diseases. Traditionally
peoples are using these on regular basis but scien tific
com mun ity not able to accepting the concep t. Scientific
evidence is needed for the purpose using these plants. The
present results also provide evidence for medicinal use of
our traditional knowledge.
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